The overall objective as elaborated in this proposal is to gain insight into the molecular mechanisms responsible for the membrane transport of electrolytes in red blood cells. There is evidence that the membrane may contain more than one type of cation pump and that certain of the so-called passive movements of cations appear to depend upon metabolism. The work is oriented toward establishing the relationship between transphosphorylation of the pump complex (and the sidedness of action of the various ligands involved) and the translocation of the transported ions. In addition, studies will also be continued in attempting to define the energy relationships and coupling mechanisms between transport and metabolism. Since certain of the metabolically coupled passive movements of cations are dependent upon the membrane potential, studies will also be directed, using our fluorescent dye technique toward defining the relevant partial membrane conductances. It is planned to correlate with the flux measurements, analyses and separate assay of various glycolytic intermediates in an attempt to identify specific substrates underlying the different metabolically coupled transports. These studies will necessarily be carried out using various ionic environments, inhibitors and metabolic conditions. It is also planned to study the binding characteristics of cardiac glycosides, K, Cs, nucleotides to intact membranes and to membrane components in an effort to define more precisely the substructure and the molecular events associated with membrane transport.